In flowering plants (angiosperms), fertilization of the egg cell by one sperm cell produces an embryo, whereas fusion of a second sperm cell with the central cell generates the endosperm. In most angiosperms like Arabidopsis, a pollen grain contains two isomorphic sperm cells required for this double fertilization process. A long-standing unsolved question is whether the two fertilization events have any preference. A tool to address this question is the usage of the cyclin-dependent kinase a1 (cdka;1) mutant pollen, which produces a single sperm-like cell (SLC). Here, we first adopt a complementation-based fluorescence-labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC. Single-cell RNA-sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell, precursor of the two sperm cells. Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2% of the ovules, single fertilization of the egg cell occurred. Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery, which fertilized the central cell, resulting in 20.7% double-fertilized ovules. This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell. Taken together, our findings have answered a long-standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis.